The M18 Aspartyl Aminopeptidase of the Human Malaria Parasite Plasmodium falciparum
2007; Elsevier BV; Volume: 282; Issue: 42 Linguagem: Inglês
10.1074/jbc.m704938200
ISSN1083-351X
AutoresFranka Teuscher, Jonathan Lowther, Tina S. Skinner‐Adams, Tobias Spielmann, Matthew W. A. Dixon, Colin M. Stack, Sheila Donnelly, Artur Mucha, Paweł Kafarski, Stamatia Vassiliou, Donald L. Gardiner, John P. Dalton, Katharine R. Trenholme,
Tópico(s)Trypanosoma species research and implications
ResumoA member of the M18 family of aspartyl aminopeptidases is expressed by all intra-erythrocytic stages of the human malaria parasite Plasmodium falciparum (PfM18AAP), with highest expression levels in rings. Functionally active recombinant enzyme, rPfM18AAP, and native enzyme in cytosolic extracts of malaria parasites are 560-kDa octomers that exhibit optimal activity at neutral pH and require the presence of metal ions to maintain enzymatic activity and stability. Like the human aspartyl aminopeptidase, the exopeptidase activity of PfM18AAP is exclusive to N-terminal acidic amino acids, glutamate and aspartate, making this enzyme of particular interest and suggesting that it may function alongside the malaria cytosolic neutral aminopeptidases in the release of amino acids from host hemoglobin-derived peptides. Whereas immunocytochemical studies using transgenic P. falciparum parasites show that PfM18AAP is expressed in the cytosol, immunoblotting experiments revealed that the enzyme is also trafficked out of the parasite into the surrounding parasitophorous vacuole. Antisense-mediated knockdown of PfM18AAP results in a lethal phenotype as a result of significant intracellular damage and validates this enzyme as a target at which novel antimalarial drugs could be directed. Novel phosphinic derivatives of aspartate and glutamate showed modest inhibition of rPfM18AAP but did not inhibit malaria growth in culture. However, we were able to draw valuable observations concerning the structure-activity relationship of these inhibitors that can be employed in future inhibitor optimization studies. A member of the M18 family of aspartyl aminopeptidases is expressed by all intra-erythrocytic stages of the human malaria parasite Plasmodium falciparum (PfM18AAP), with highest expression levels in rings. Functionally active recombinant enzyme, rPfM18AAP, and native enzyme in cytosolic extracts of malaria parasites are 560-kDa octomers that exhibit optimal activity at neutral pH and require the presence of metal ions to maintain enzymatic activity and stability. Like the human aspartyl aminopeptidase, the exopeptidase activity of PfM18AAP is exclusive to N-terminal acidic amino acids, glutamate and aspartate, making this enzyme of particular interest and suggesting that it may function alongside the malaria cytosolic neutral aminopeptidases in the release of amino acids from host hemoglobin-derived peptides. Whereas immunocytochemical studies using transgenic P. falciparum parasites show that PfM18AAP is expressed in the cytosol, immunoblotting experiments revealed that the enzyme is also trafficked out of the parasite into the surrounding parasitophorous vacuole. Antisense-mediated knockdown of PfM18AAP results in a lethal phenotype as a result of significant intracellular damage and validates this enzyme as a target at which novel antimalarial drugs could be directed. Novel phosphinic derivatives of aspartate and glutamate showed modest inhibition of rPfM18AAP but did not inhibit malaria growth in culture. However, we were able to draw valuable observations concerning the structure-activity relationship of these inhibitors that can be employed in future inhibitor optimization studies. It is estimated that 3.2 billion people currently live in areas where there is a risk of malaria transmission. Three to five hundred million of these individuals become infected each year and over two million die (1Breman J. Am. J. Trop. Med. Hyg. 2001; 64: 1-11Crossref PubMed Scopus (688) Google Scholar). The groups most affected by malaria are children under five years of age and pregnant women in sub-Saharan Africa. Parasite resistance to most of the currently used antimalarial drugs is now widespread and resistance to new drugs is developing (2Jambou R. Legrand E. Niang M. Khim N. Lim P. Volney B. Ekala M.T. Bouchier C. Esterre P. Fandeur T. Mercereau-Puijalon O. Lancet. 2005; 9501: 1960-1963Abstract Full Text Full Text PDF Scopus (420) Google Scholar). With an effective vaccine at least 15 years away there is an urgent need for new malaria treatments. During the intraerythrocytic phase of development the parasite digests 65-75% of the host cell hemoglobin. A proportion of this digested hemoglobin (16%) is used for protein synthesis (3Krugliak M. Zhang J. Ginsburg H. Mol. Biochem. Parasitol. 2002; 119: 249-256Crossref PubMed Scopus (166) Google Scholar). Hemoglobin degradation is also important in reducing the colloid-osmotic pressure within the infected erythrocyte, which prevents premature cell lysis during parasite growth and establishes a concentration gradient by which rare amino acids enter the malaria-infected erythrocyte from host serum (4Lew V.L. Macdonald L. Ginsburg H. Krugliak M. Tiffert T. Blood Cells Mol. Dis. 2004; 32: 353-359Crossref PubMed Scopus (77) Google Scholar, 5Goldberg D.E. Curr. Top. Microbiol. Immunol. 2005; 295: 275-291Crossref PubMed Scopus (173) Google Scholar, 6Becker K. Kirk K. Trends Parasitol. 2004; 20: 590-596Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The degradation of hemoglobin to peptide fragments occurs within a specialized acidic digestive vacuole (DV) 9The abbreviations used are: DV, digestive vacuole; NHMec, 7-amido-4-methylcoumarin; phosphorus containing inhibitors: 1, 3-amino-3-phosphonopropionic acid; 2, 3-amino-3-(P-methylphosphinyl)propionic acid; 3, 3-amino-3-[P-(2-carboxypropyl)phosphinyl]propionic acid; 4, 4-amino-4-phosphonobutyric acid; 5, 4-amino-4-(P-phenylphosphinyl)butyric acid; 6, 4-amino-4-[P-(2-carboxypropyl)phosphinyl]butyric acid; 7, 1-amino-1,4-butanediphosphonic acid; 8, 5-amino-5-phosphonopentanoic acid; M1MAA, M1 membrane alanyl aminopeptidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high pressure liquid chromatography; Mes, 4-morpholineethanesulfonic acid; PVM, parasitophorous vacuole membrane; PfM18APP, Plasmodium falciparum aspartyl aminopeptidase; M17LAP, membrane 17 leucine aminopeptidase. by the action of aspartic, cysteine, and metalloendoproteases and by dipeptidases. However, small peptide fragments are transported from the DV to the parasite cytosol where they are degraded into free amino acids by amino- and carboxypeptidases (5Goldberg D.E. Curr. Top. Microbiol. Immunol. 2005; 295: 275-291Crossref PubMed Scopus (173) Google Scholar). There are eight aminopeptidases within the genome of the most clinically significant malaria species, Plasmodium falciparum (www.plasmodb.org): four methionine aminopeptidases, two neutral aminopeptidases (leucine aminopeptidase (M17LAP) and membrane alanine aminopeptidase (M1MAA)), a prolyl aminopeptidase (PAP), and an aspartyl aminopeptidase (M18AAP). Using specific enzyme inhibitors, one of the methionine aminopeptidases has been validated in vitro and in vivo as a potential drug target (7Chen X. Chong C.R. Shi L. Yoshimoto T. Sullivan Jr., D.J. Liu J.O. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14548-14553Crossref PubMed Scopus (88) Google Scholar) and inhibitors of the M1MAA and M17LAP have been shown to prevent malaria growth in culture (8Nankya-Kitaka M.F. Curley G.P. Gavigan C.S. Bell A. Dalton J.P. Parasitol. Res. 1998; 84: 552-558Crossref PubMed Scopus (80) Google Scholar). Therefore, other aminopeptidases may also prove to be good targets for new antimalarial agents, particularly as part of drug combinations. Aspartyl aminopeptidases are members of the M18 family of metalloproteases (www.merops.sanger.ac.uk). Unlike the methionine and neutral aminopeptidases, few aspartyl aminopeptidases (M18AAP) have been characterized; at present, there is only limited information reported for the aspartyl aminopeptidase of mammals (9Wilk S. Wilk E. Magnusson R.P. J. Biol. Chem. 1998; 273: 15961-15970Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), yeast (10Yokoyama R. Kawasaki H. Hirano H. FEBS J. 2006; 273: 192-198Crossref PubMed Scopus (32) Google Scholar), and bacteria. 10T. Min and L. Shapiro, unpublished data. The lack of available substrate and inhibitor reagents has contributed to our poor understanding of the function of these enzymes. However, because of their restricted specificities for the N-terminal acidic amino acids, aspartic and glutamic acid, which cannot be cleaved by any other aminopeptidases, they are thought to act in concert with other aminopeptidases to facilitate protein turnover. In humans, a more specific function in the conversion of angiotensin II to the vasoactive angiotensin III within the brain has been implicated (12Wright J.W. Harding J.W. Brain Res. Brain Res. Rev. 1997; 25: 96-124Crossref PubMed Scopus (242) Google Scholar). Here, we report for the first time the physicobiochemical properties, cellular expression, and distribution of the P. falciparum aspartyl aminopeptidase (PfM18AAP). We have produced a functionally active recombinant form of the enzyme that exhibits comparable properties to the native form measured in malaria cytosolic extracts. Our studies show that the PfM18AAP is expressed in the parasite cytosol and exported to the parasitophorous vacuole of the parasite indicating that whereas the enzyme may function in the final stages of hemoglobin digestion it may also have an additional function outside the parasite. Antisense-mediated inhibition of the PfM18AAP results in a lethal phenotype as a result of significant morphological changes to the parasite and, therefore, pinpoints the enzyme as a promising target for new anti-malarial drug development. However, novel inhibitors of aspartyl aminopeptidases that exhibit modest activity against the native and recombinant PfM18AAP do not prevent the growth of the parasites in culture. Parasites and Preparation of Parasite Extracts—P. falciparum clone D10 was cultured as described (13Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6184) Google Scholar). For experiments investigating the stage-specific expression of PfM18AAP, parasites were synchronized using two rounds of sorbitol treatment (14Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2839) Google Scholar), and parasites harvested at ring, trophozoite, and schizont stages. After washing infected red blood cells in PBS, parasites were released by incubation with either (1Breman J. Am. J. Trop. Med. Hyg. 2001; 64: 1-11Crossref PubMed Scopus (688) Google Scholar) 0.03% saponin/PBS on ice or (2Jambou R. Legrand E. Niang M. Khim N. Lim P. Volney B. Ekala M.T. Bouchier C. Esterre P. Fandeur T. Mercereau-Puijalon O. Lancet. 2005; 9501: 1960-1963Abstract Full Text Full Text PDF Scopus (420) Google Scholar) with 600 units/ml streptolysin O/PBS at 37 °C. Resulting parasite pellets were washed three times with PBS, re-suspended in 100 μl of PBS, and extracted by two cycles of freezethaw at -80 °C followed by centrifugation at 14,000 × g. Supernatants were stored at -20 °C. Membrane preparations of infected red blood cells were produced by hypotonic lysis followed by centrifugation at 5,000 × g (15Spielmann T. Gardiner D.L. Beck H.P. Trenholme K.R. Kemp D.J. Mol. Microbiol. 2006; 59: 779-794Crossref PubMed Scopus (65) Google Scholar). The crude membrane pellets were treated with 0.5 ml of 0.1 m sodium carbonate or 1 ml of 1% Triton X-100 and separated into pellet and supernatant by centrifugation at 16,000 × g. Proteins in the supernatant were concentrated by precipitation with trichloroacetic acid (10% final concentration). Production, Purification, and Characterization of Functionally Active Recombinant Aspartyl Aminopeptidase—The PlasmoDB annotated gene sequence PFI1570c encoding the putative PfM18AAP was chemically synthesized by GENEART GmbH (GeneArt, Germany) using codons for optimized gene expression in the yeast Pichia pastoris. As reported by Stack et al. (16Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) for the M17 leucine aminopeptidase, the malaria PfM18AAP gene with the codons optimized for P. pastoris was successfully expressed in a functional form in insect cells. Potential N-linked glycosylation sites were removed in gene synthesis by replacing the asparagine of all Asn-X-Thr/Ser with Gln. This construct was recombined with BaculoDirect™ C-terminal linear DNA (Invitrogen) and transfected into Sf9 (Spodoptera frugiperda) cells (16Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Like the M17 leucine aminopeptidase, the malaria PfM18AAP gene while synthesized in the codon style of P. pastoris, was not able to be expressed successfully in yeast (16Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). For protein expression, Sf9 insect cells were infected at the cell density 3 × 106 cells/ml with PfM18AAP recombinant Baculovirus at a multiplicity of infection of 2-5 plaque forming units/cell. rPfM18AAP, which was expressed bearing a His6 tag, was isolated from insect cells by affinity chromatography on a Ni-NTA-agarose column as previously described (16Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The purity and molecular size of isolated rPfM18AAP was analyzed using 12% reducing SDS-PAGE and gel filtration HPLC on a Superdex-200 column using a Pharmacia Biotech Smart System. The mobile phase was PBS, the column was run at a flow rate of 40 μl per min, and 40-μl fractions were collected. The activity and substrate specificity of purified rPfM18AAP was determined by measuring initial rates of hydrolysis of the fluorogenic peptide substrates H-Asp-NHMec and H-Glu-NHMec at an excitation wavelength of 370 nm and an emission wavelength of 460 nm using a Bio-Tek KC4 microfluorimeter (16Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). rPfAAP (60 nm) was incubated in 50 mm Tris-HCl, pH 7.5, for 30 min before addition to the substrate. The pH profile for rPfM18AAP activity was determined from the initial rates of H-Asp-NHMec hydrolysis carried out in constant ionic strength (I = 0.1) with acetate/Mes/Tris buffers, pH 4-11 (17Ellis K.J. Morrison J.F. Methods Enzymol. 1982; 87: 405-426Crossref PubMed Scopus (649) Google Scholar). The pH stability was determined by incubating rPfM18AAP in these buffers for 1 h at 37 °C before assaying for residual activity at pH 7.5. To investigate the effect of metal ions on enzymatic kinetic parameters, rPfM18AAP was incubated with various metal ions for 30 min prior to initiation of the enzymatic reaction. Initial rates were obtained at 37 °C over a range of H-Asp-NHMec substrate concentrations spanning Km (0.2-500 μm) and at fixed enzyme concentrations. The effect of bestatin, metal chelators, and dithiothreitol on rPfM18AAP activity was investigated by measuring the initial rate of hydrolysis of 25 μm H-Asp-NHMec at pH 7.5 in the presence of each compound. Each rate was compared with the control rate containing only enzyme and substrate. M18AAP activity in parasite extracts were determined by first incubating aliquots of the extract in 50 mm Tris-HCl, pH 7.5, containing 1 mm CoCl2 for 20 min before addition to 25 μm H-Asp-NHMec. Leucyl aminopeptidase activity in the extracts was measured using 10 μm H-Leu-NHMec as described (16Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Phosphorus Containing Inhibitors—A series of α-phosphonic (compounds 1, 4, 7, and 8) and α-phosphinic (2 and 5) analogues of acidic amino acids (18Kafarski, P., and Zon, J. (2000) in Aminophosphonic and Aminophosphinic Acids, Chemistry and Biological Activity (Kukhar, V. P., and Hudson, H. R., eds) pp. 33-74, John Wiley & Sons, Chichester, United KingdomGoogle Scholar), as well as phosphinate dipeptides (3 and 6) (19Georgiadis D. Matziari M. Vassiliou S. Dive V. Yiotakis A. Tetrahedron. 1999; 55: 14635-14648Crossref Scopus (40) Google Scholar) were tested for their inhibitory activity toward PfM18AAP using the fluorogenic peptide assay described above. Polyclonal Antibody Production and Immunoblotting Analysis—Polyclonal antiserum was prepared against a 15-mer peptide, C*FSHKENSQNKRDDQ, corresponding to amino acid residues 211-224 of the putative P. falciparum aspartyl aminopeptidase (PFI1570c) as described previously (20Gardiner D.L. Spielmann T. Dixon M.W.A. Hawthorne P.L. Ortega M.R. Anderson K.L. Skinner-Adams T.S. Kemp D.J. Trenholme K.R. Parasitol. Res. 2004; 93: 64-67Crossref PubMed Scopus (19) Google Scholar). Proteins of saponin-lysed parasite extracts were resolved on reducing 10% SDS-PAGE gels, transferred to a nitrocellulose membrane, and probed with the anti-PfM18AAP antisera (1:250 dilution) followed by a horseradish peroxidase-labeled anti-mouse IgG antibody (1:5000 dilution, Chemicon International Inc.) (21Hawthorne P.L. Trenholme K.R. Skinner-Adams T.S. Spielmann T. Fischer K. Dixon M.W.A. Ortega M.A. Anderson K.L. Kemp D.J. Gardiner D.L. Mol. Biochem. Parasitol. 2004; 136: 181-189Crossref PubMed Scopus (78) Google Scholar). The membrane was stripped and re-probed with an anti-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) rabbit antibody (1:5000 dilution) to demonstrate equal loading and transfer of malaria proteins (22Daubenberger C.A. Tisdale E.J. Curcic M. Diaz D. Silvie O. Mazier D. Eling W. Bohrmann B. Matile H. Pluschke G. Biol. Chem. 2003; 384: 1227-1237Crossref PubMed Scopus (58) Google Scholar). Fluorescence Microscopy—Fluorescence and phase-contrast images were collected with an Axioscope 2 Mot+ (Zeiss) equipped with a Zeiss ×63/1.4 Plan Apochromat lens. Live parasites were mounted in PBS and observed at ambient temperature. Parasite DNA was visualized by adding Hoechst dye (0.5 μg/ml) and incubating at 37 °C for 10 min prior to mounting. For indirect fluorescence, concanavalin A (0.5 mg/ml) was added to each well of a multiwell slide and incubated for 30 min at 37 °C after which infected red blood cells were added, incubated at room temperature for 15 min, and unbound cells were removed by washing with PBS. The cells were fixed in 4% formaldehyde, 0.005% glutaraldehyde and probed with anti-PfM18AAP antiserum or with a mouse monoclonal antibody to GFP or c-myc (all diluted 1:500). Bound antibody was visualized with goat anti-mouse Ig-Cy2 (10 μg/ml). Transmission Electron Microscopy—Infected red blood cells were fixed with 3% glutaraldehyde in cacodylate buffer, pH 7.2, and processed according to standard methods (23Glauert, A. M. (1974) in Practical Methods in Electron Microscopy, Part I (Glauert, A. M., ed) Vol. 3, pp. 1-207, North-Holland Publishing Co., AmsterdamGoogle Scholar). After embedding in Spurr low viscosity resin, ultrathin sections (∼50-60 nm thick) were prepared and stained with uranyl acetate and lead citrate and examined with a JEOL 1200EX transmission electron microscope operating at 80 kV. Northern Blotting—Northern blotting was performed with total RNA extracts prepared using TRIzol (Invitrogen) (21Hawthorne P.L. Trenholme K.R. Skinner-Adams T.S. Spielmann T. Fischer K. Dixon M.W.A. Ortega M.A. Anderson K.L. Kemp D.J. Gardiner D.L. Mol. Biochem. Parasitol. 2004; 136: 181-189Crossref PubMed Scopus (78) Google Scholar). Blots were probed with a purified 1713-bp PCR fragment corresponding to the full-length CDS of the aspartyl aminopeptidase (PFI1570c) amplified from genomic P. falciparum DNA using primers PFI1570ASF (ctgcagatggataagaaagctagggaa) and PFI1570ASR (agatcttttgtcgtggacacatgtgga). Probes were labeled with [α-32P]dATP by random priming (DECAprime II, Ambion Inc). The probe was hybridized overnight at 40 °C in a hybridization buffer containing formamide (Northern Max; Ambion). The filter was washed once at low stringency and twice at high stringency (Northern Max; Ambion), then exposed overnight. For specific sense and antisense probes, 26 ng of the above purified PCR product was primed with either a 3′ primer (acttcgctaagagatcctatt) to generate a probe that specifically binds to the endogenous mRNA or a 5′ primer (atggttcgatagaagtttagg) to produce a probe that binds specifically to the transgenic antisense RNA. Geometric amplification of the PCR product was performed using Taq polymerase under the following cycling conditions: denaturing at 94 °C for 30 s, annealing at 50 °C for 30 s, followed by extension at 68 °C for 2 min, for 30 cycles. These probes were hybridized to blots containing RNA extracted from both D10 wild-type parasites and transgenic cultures. Construction of the Transgenic Expression Plasmids—PFI1570c was amplified from P. falciparum clone D10 genomic DNA. The forward primer for PFI1570c was PFI1570F (agatctatggataagaaagctagggaa) and contained a BglII restriction site (in bold). The reverse primer was PFI1570R (ctgcagtttgtcgtggacacatgtgga) and contained a PstI site (in bold). Antisense primers were designed with restriction sites in the reverse orientation (PstI forward and BglII reverse). The PCR products were cloned into pGEM using a TA cloning system (Promega) and sequenced to confirm that no Taq-associated errors had occurred. In the case of GFP and c-myc constructs full-length fragments were digested out of the pGEM vector using BglII and PstI and subcloned into the Gateway™ compatible entry vectors pHGFPB and pHcmycB (Gateway, Invitrogen) that had previously been digested using BglII and PstI. Either a GFP tag or a c-myc tag were ligated in-frame at the 3′ end of the introduced gene sequence, respectively (24Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Stack C.M. Dalton J.P. J. Biol. Chem. 2006; 281: 1741-1745Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These introduced genes were under the control of the HSP86 promoter. The antisense sequence was cloned into the pHcmycB vector. These entry vectors were designated pHB-PFI1570c-GFP and pHB-PFI1570c-cmyc, respectively, whereas the antisense plasmid was designated pHB-PFI1570-AS. Using those entry vectors and Gateway™ compatible destination vectors with a destination cassette and a second cassette containing the human dihydrofolate reductase synthase gene under the control of the P. falciparum calmodulin promoter as a selectable marker, clonase reactions were then performed. These final plasmids were designated pHH1-PFI1570c-GFPB (GFP tag), pHH1-PFI1570c-cmycB (c-myc tag), and pHH1-PFI1570c-AS (antisense). For transfection, ring stage parasites were subjected to electroporation in the presence of 50 μg of plasmid DNA as described (25Spielmann T. Dixon M.W.A. Hernandez-Valladares M. Hannemann M. Trenholme K.R. Gardiner D.L. Int. J. Parasitol. 2006; 36: 1245-1248Crossref PubMed Scopus (10) Google Scholar). Parasites resistant to WR99210 were obtained up to 25 days later. Sequence Analysis—Aspartyl aminopeptidase proteases from various species were retrieved from the NCBI data base. BLAST hits were aligned using CLUSTAL W at PBIL and alignments drawn using ESPrit 2.0. Interrogation of PlasmoDB Discovered a Putative Aspartyl Aminopeptidase—PFI1570c, the putative aspartyl aminopeptidase of P. falciparum is located on chromosome 9 and as annotated by PlasmoDB consists of a single open reading frame of 1713 bp that translates into a protein of 570 amino acids. PFI1570c contains two signature domains, an N-terminal aspartyl aminopeptidase domain (LAP4: residues 1-186, e value 5e-34), and a C-terminal aminopeptidase 1 zinc metalloprotease M18 domain (Peptidase_18: residues 246-556, Pfam0217; e value 2e-47). The overall sequence identity between the aspartyl aminopeptidases (M18AAP) of the various rodent Plasmodium sp. is 61-65% and would be in the region of 83-88% only for the fact that PfM18AAP possesses two unique sequence insertions. These insertions, which are predicted to form loops, are present in the central portions of the enzymes and clearly do not prevent the enzyme from forming an octomeric structure (see below). Whereas low levels of identity exists between the PfM18AAP and the human (31%) and yeast M18 aspartyl aminopeptidases (27%) the three histidine residues (His-94, His-170, and His-440) that are predicted from site-directed mutagenesis studies to be critical for enzymatic activity and another (His-352) essential for stabilization of quaternary structure of human M18AAP are conserved and are indicated in the alignment presented in supplementary materials Fig. S1. Biochemical Characterization of Functionally Active Recombinant rPfM18AAP—Recombinant rPfM18AAP was purified from Baculovirus-transformed insect cells by affinity chromatography on a Ni-NTA resin and resolved as a single protein of ∼65 kDa in reducing SDS-PAGE (Fig. 1A). Purified rPfM18AAP efficiently cleaved the simple fluorogenic substrates H-Asp-NHMec and H-Glu-NHMec with kcat/Km values of 129.9 and 82.3 m-1 s-1, respectively (Table 1). The purified enzyme exhibited aminopeptidase activity against the fluorogenic substrate H-Asp-NHMec between pH 6.0 and 9.0 with optimal activity at pH 7.5 (Fig. 1B). The enzyme was stable when stored for 1 h at 37°C over the pH range 6 to 11. No hydrolysis was observed against the fluorogenic substrates H-Leu-NHMec, H-Phe-NHMec, H-Ala-NHMec, H-Pro-NHMec, H-Gly-NHMec, H-Val-NHMec, H-Arg-NHMec, and H-Ile-NHMec when the substrate concentration was 100 μm.TABLE 1Kinetic parameters for the hydrolysis of H-Asp-NHMec and H-Glu-NHMec by rPfM18AAP at pH 7.5 in the presence and absence of cobaltSubstrate1 mm CoKmkcatkcat/Kmμms-1m-1 s-1Asp-NHMec-384.8 ± 32.10.050 ± 0.002129.9Asp-NHMec+327.3 ± 2.20.354 ± 0.0081081.6Glu-NHMec-135.1 ± 15.60.011 ± 0.000482.3Glu-NHMec+136.3 ± 8.70.33 ± 0.0062421.1 Open table in a new tab rPfM18AAP activity was reduced to 14 and 8.3% after incubating with 10 mm EDTA and 20 mm o-phenanthroline, respectively, demonstrating that metal ions are necessary for enzyme activity (data not shown). A study of the effect of various metal ions on the activity of the enzyme showed that it was enhanced by Co(II), but not by Ca(II), Fe(II), Mg(II), Mn(II), and Ni(II), whereas Zn(II) at a concentration of 1 mm abolished enzyme activity (data not shown). When the enzyme was incubated for 30 min in 50 mm Tris-HCl, pH 7.5, and containing 1 mm Co(II) the kcat/Km values of the enzyme for substrates H-Asp-NHMec and H-Glu-NHMec increased ∼8- and 30-fold, respectively (Table 1). These enzyme kinetics studies showed that kcat values increased while the Km values remained unchanged, which indicates that the metal ion does not affect binding of the substrate but does increase the catalytic efficiency of the enzyme. PfM18AAP Activity in Soluble Extracts of Malaria Parasites—Activity against the M18AAP-specific substrates H-Asp-NHMec and H-Glu-NHMec was detected in soluble extracts of malaria parasites and had similar characteristics to the activity of purified rPfM18AAP. The Km values with parasite extracts of 216 and 251 μm for H-Asp-NHMec and H-Glu-NHMec, respectively, are similar to the Km values of 327 and 136 μm obtained with rPfM18AAP for the same substrates (Table 1). The specific activity (μmol of NHMec released/min/mg of protein) for the enzyme in these extracts was 662.5 and 607 for H-Asp-NHMec and H-Glu-NHMec, respectively. Furthermore, the activity against these substrates was enhanced almost 10-fold by Co(II) but was not enhanced by other metal ions studied (data not shown). The molecular size of the rPfM18AAP and native enzyme in parasite extracts were determined by HPLC size chromatography by assaying fractions for activity toward H-Asp-NHMec. H-Asp-NHMec-cleaving activity in parasite extracts eluted from the HPLC molecular size column at the same retention time as the activity of purified rPfM18AAP corresponding to a molecular size of 560 kDa (Fig. 1C). The data suggest that both native and recombinant PfM18AAP possess an octomeric structure consistent with that observed for human M18AAP (26Wilk S. Wilk E. Magnusson R.P. Arch. Biochem. Biophys. 2002; 407: 176-183Crossref PubMed Scopus (27) Google Scholar). PfM18AAP Is Transcribed and Translated in Intra-erythrocytic Stage Parasites—Northern blot analysis showed that wild type D10 parasites transcribed a single species of mRNA with an apparent size of ∼3 kb when hybridized using a 1713-bp fragment comprising the complete CDS of the PfM18AAP (Fig. 2A). PfM18AAP appeared to be most abundantly expressed in ring stage parasites (Fig. 2A). Western blot analysis showed that when saponin-lysed parasite extracts of the same cultures were probed with anti-PfM18AAP antiserum a single protein species with an apparent molecular mass of 65 kDa was observed, which corresponded closely with the theoretical molecular mass of 65.6 kDa (Fig. 2B). Immunoblot analysis of soluble, peripheral membrane-bound and integral m
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